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Protein complexes and proteolytic activation of the cell wall hydrolase RipA regulate septal resolution in mycobacteria.

Chao MC, Kieser KJ, Minami S, Mavrici D, Aldridge BB, Fortune SM, Alber T, Rubin EJ - PLoS Pathog. (2013)

Bottom Line: Peptidoglycan hydrolases are a double-edged sword.They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria.Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America.

ABSTRACT
Peptidoglycan hydrolases are a double-edged sword. They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria. How bacteria ensure that peptidoglycan hydrolases function only in the correct spatial and temporal context remains largely unknown. Here, we demonstrate that dysregulation converts the essential mycobacterial peptidoglycan hydrolase RipA to an autolysin that compromises cellular structural integrity. We find that mycobacteria control RipA activity through two interconnected levels of regulation in vivo-protein interactions coordinate PG hydrolysis, while proteolysis is necessary for RipA enzymatic activity. Dysregulation of RipA protein complexes by treatment with a peptidoglycan synthase inhibitor leads to excessive RipA activity and impairment of correct morphology. Furthermore, expression of a RipA dominant negative mutant or of differentially processed RipA homologues reveals that RipA is produced as a zymogen, requiring proteolytic processing for activity. The amount of RipA processing differs between fast-growing and slow-growing mycobacteria and correlates with the requirement for peptidoglycan hydrolase activity in these species. Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.

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Model for RipA activation through proteolytic processing and protein interactions.RipA is produced as a full length zymogen with an N-terminal secretion signal. Once in the periplasm, RipA can bind to its interaction partners, leading to recruitment of proteases that cleave the extended loop between an N-terminal blocking domain and the C-terminal peptidoglycan hydrolase domain. The cleaved RipA C-terminal domain is now active and functions by cleaving the septal peptidoglycan, splitting the daughter cells, perhaps in conjunction with other hydrolases.
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ppat-1003197-g008: Model for RipA activation through proteolytic processing and protein interactions.RipA is produced as a full length zymogen with an N-terminal secretion signal. Once in the periplasm, RipA can bind to its interaction partners, leading to recruitment of proteases that cleave the extended loop between an N-terminal blocking domain and the C-terminal peptidoglycan hydrolase domain. The cleaved RipA C-terminal domain is now active and functions by cleaving the septal peptidoglycan, splitting the daughter cells, perhaps in conjunction with other hydrolases.

Mentions: Together, our work demonstrates that RipA regulation occurs at multiple levels post-transcriptionally. We did not see any transcriptional downregulation in cells transitioning into non-replicating conditions (Figure S4). In fact, there was a significant increase in RipA transcription during the transition to stationary phase, but the functional consequence of this observation remains unknown. Furthermore, while overexpression of the dominant negative RipASm C408A allele modulates processing of endogenous RipA (Fig S6A), this is due to competition for processing and not transcriptional feedback, even at high induction conditions (Figure S6B). This lack of transcriptional modulation is consistent with the observation that ripA expression has only limited variation across dozens of published experimental conditions (summarized on TBDB [31]), including general and antibiotic stresses. The only conditions under which ripA expression has been found to change are under non-replication conditions and, recently, when cells are blocked in cell division [32]. In the latter work, Plocinska et al found that ripA can be regulated by the MtrAB two-component system. Specifically, inhibiting septum formation prevents MtrB, which localizes at the septum, from activating the MtrA response regulator, leading to ripA downregulation. The authors proposed an interesting model in which ripA transcription could be upregulated by MtrB when it assembles at the division site; however, it remains unclear whether MtrAB regulation of ripA transcription occurs during normal growth or, instead, represents a stress response when cell division is inhibited. While the question of ripA transcriptional regulation during vegetative growth remains to be tested, our work suggests that post-translational mechanisms like processing may represent a key way of controlling RipA hydrolytic activity during growth. Therefore, we propose that protein-protein interactions help establish RipA function at the septum, where it is then aided in becoming proteolytically cleaved in the periplasm (Figure 8). After enzymatic activation, functional RipA can rely on both upstream and downstream protein interactions to help place it in the correct context during cell division—inhibition of cognate PG synthases can lead to dysregulated cell wall hydrolysis. The benefit of having multiple levels of RipA regulation is that the cell can exert a tighter control over RipA's activation and potential autolysin activity.


Protein complexes and proteolytic activation of the cell wall hydrolase RipA regulate septal resolution in mycobacteria.

Chao MC, Kieser KJ, Minami S, Mavrici D, Aldridge BB, Fortune SM, Alber T, Rubin EJ - PLoS Pathog. (2013)

Model for RipA activation through proteolytic processing and protein interactions.RipA is produced as a full length zymogen with an N-terminal secretion signal. Once in the periplasm, RipA can bind to its interaction partners, leading to recruitment of proteases that cleave the extended loop between an N-terminal blocking domain and the C-terminal peptidoglycan hydrolase domain. The cleaved RipA C-terminal domain is now active and functions by cleaving the septal peptidoglycan, splitting the daughter cells, perhaps in conjunction with other hydrolases.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585148&req=5

ppat-1003197-g008: Model for RipA activation through proteolytic processing and protein interactions.RipA is produced as a full length zymogen with an N-terminal secretion signal. Once in the periplasm, RipA can bind to its interaction partners, leading to recruitment of proteases that cleave the extended loop between an N-terminal blocking domain and the C-terminal peptidoglycan hydrolase domain. The cleaved RipA C-terminal domain is now active and functions by cleaving the septal peptidoglycan, splitting the daughter cells, perhaps in conjunction with other hydrolases.
Mentions: Together, our work demonstrates that RipA regulation occurs at multiple levels post-transcriptionally. We did not see any transcriptional downregulation in cells transitioning into non-replicating conditions (Figure S4). In fact, there was a significant increase in RipA transcription during the transition to stationary phase, but the functional consequence of this observation remains unknown. Furthermore, while overexpression of the dominant negative RipASm C408A allele modulates processing of endogenous RipA (Fig S6A), this is due to competition for processing and not transcriptional feedback, even at high induction conditions (Figure S6B). This lack of transcriptional modulation is consistent with the observation that ripA expression has only limited variation across dozens of published experimental conditions (summarized on TBDB [31]), including general and antibiotic stresses. The only conditions under which ripA expression has been found to change are under non-replication conditions and, recently, when cells are blocked in cell division [32]. In the latter work, Plocinska et al found that ripA can be regulated by the MtrAB two-component system. Specifically, inhibiting septum formation prevents MtrB, which localizes at the septum, from activating the MtrA response regulator, leading to ripA downregulation. The authors proposed an interesting model in which ripA transcription could be upregulated by MtrB when it assembles at the division site; however, it remains unclear whether MtrAB regulation of ripA transcription occurs during normal growth or, instead, represents a stress response when cell division is inhibited. While the question of ripA transcriptional regulation during vegetative growth remains to be tested, our work suggests that post-translational mechanisms like processing may represent a key way of controlling RipA hydrolytic activity during growth. Therefore, we propose that protein-protein interactions help establish RipA function at the septum, where it is then aided in becoming proteolytically cleaved in the periplasm (Figure 8). After enzymatic activation, functional RipA can rely on both upstream and downstream protein interactions to help place it in the correct context during cell division—inhibition of cognate PG synthases can lead to dysregulated cell wall hydrolysis. The benefit of having multiple levels of RipA regulation is that the cell can exert a tighter control over RipA's activation and potential autolysin activity.

Bottom Line: Peptidoglycan hydrolases are a double-edged sword.They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria.Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America.

ABSTRACT
Peptidoglycan hydrolases are a double-edged sword. They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria. How bacteria ensure that peptidoglycan hydrolases function only in the correct spatial and temporal context remains largely unknown. Here, we demonstrate that dysregulation converts the essential mycobacterial peptidoglycan hydrolase RipA to an autolysin that compromises cellular structural integrity. We find that mycobacteria control RipA activity through two interconnected levels of regulation in vivo-protein interactions coordinate PG hydrolysis, while proteolysis is necessary for RipA enzymatic activity. Dysregulation of RipA protein complexes by treatment with a peptidoglycan synthase inhibitor leads to excessive RipA activity and impairment of correct morphology. Furthermore, expression of a RipA dominant negative mutant or of differentially processed RipA homologues reveals that RipA is produced as a zymogen, requiring proteolytic processing for activity. The amount of RipA processing differs between fast-growing and slow-growing mycobacteria and correlates with the requirement for peptidoglycan hydrolase activity in these species. Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.

Show MeSH
Related in: MedlinePlus